番茄环纹斑点病毒N和NSm基因的VIGS载体构建

Construction of VIGS vector of tomato zonate spotted tospovirus N and NSm gene

【目的】番茄环纹斑点病毒(Tomato zonate spot orthotospovirus,TZSV)由传毒介体蓟马以持久增殖型方式传播,且能与其他病毒复合侵染作物,对中国西南地区重要蔬菜的产量和观赏作物的品质造成严重影响,给当地农业生产带来了严重危害。TZSV的N和NSm基因与病毒分类、运动和系统侵染密切相关。本研究通过构建N和NSm基因沉默载体,以期为进一步研究TZSV N和NSm基因在病毒侵染方面的功能提供材料,为抗病育种以及田间绿色防控提供一些依据。【方法】根据TZSV N和NSm基因序列设计特异性引物,利用RT-RCR技术扩增得到TZSV N和NSm基因片段,将扩增得到的目的基因连接到pEASY-Blunt-Zero载体上,含有N和NSm基因序列的pEASY-Blunt-Zero载体和pTRV-pTV00载体用Bam HI和Hind III限制性内切酶进行双酶切,并将酶切产物进行纯化回收,纯化后产物与pTRV-pTV00载体使用T4 DNA连接酶连接,获得pTRV-PTV00-N和pTRV-PTV00-NSm重组载体,将其转入根癌农杆菌GV3101中,并注射到本氏烟和栽培烟K326中,通过实时荧光定量PCR(RT-qPCR)检测病毒N和NSm基因的沉默效率,间接酶联免疫吸附试验(ID-ELISA)检测N和NSm蛋白的含量。【结果】RT-qPCR分析表明,本生烟和栽培烟K326中N和NSm基因在接种TZSV后5 d沉默效率均大于90%。ID-ELISA结果显示,2个蛋白含量在不同时间内均显著下降,其中NSm蛋白含量连续9 d显著下降。【结论】TZSV N和NSm基因沉默载体构建成功,并成功试验于本氏烟和栽培烟K326。通过构建TZSV N和NSm基因沉默载体,为研究TZSV致病机理提供了材料,为田间抗TZSV育种和防控提供了理论依据。

【Objective】Tomato zonate spot orthosporospirus(TZSV)are transmitted in a persistent way by thrips,which has seriously affected the yield and quality of important vegetables and ornamental crops in Southwest China,and has brought serious harm to local agricultural production.The N and NSm genes of TZSV are closely related to virus classification,movement and systemic infection.The study aims to provide materials for further research on the function of TZSV N and NSm genes in virus infection,and provide some basis for disease resistance breeding and green control in the field based on constructing N and NSm gene silencing vectors.【Method】In the study,specific primers were designed according to the sequences of TZSV N and NSm genes,N and NSm gene fragments were amplified by RT-RCR and were cloned into the vector of pEASY-Blunt-Zero.The pEASY-Blunt-Zero vector containing N and NSm gene sequences and pTRV-pTV00 vector were both digested with Bam HI and Hind III restriction enzymes and were purified and recycled,thereafter the purified products were linked to pTRV-pTV00 vector using T4 DNA ligase to obtain pTRV-PTV00-N and pTRV-PTV00-NSm recombinant vectors,which were then transferred into Agrobacterium tumefaciens GV3101 and injected into Nicotiana benthamiana and N.tabacum cv.K326.The silencing efficiency of N and NSm genes was detected by real-time fluorescent quantitative PCR(RT-qPCR),and the content of N and NSm proteins was detected by indirect enzyme-linked immunosorbent assay(ID-ELISA).【Result】RT-qPCR analysis showed that the silencing efficiency of N and NSm genes in N.benthamiana and N.tabacum cv.K326 was greater than 90%5 days after TZSV inoculation.The results of ID-ELISA showed that the content of two proteins decreased significantly in different time,and the content of NSm protein decreased significantly for 9 consecutive days.【Conclusion】The construction of N and NSm gene silencing vectors was successful.The experiment was successfully conducted on N.benthamiana and N.tabacum cv.K326.The construction of N and NSm gene silencing vectors provides materials for studying the pathogenesis of TZSV,and provides a theoretical basis for breeding and control of TZSV resistance in the field.